PROJECT SUMMARY In women, aging is characterized by a marked decrease in estrogen secretion, which has been shown to negatively impact many physiological systems and contribute to the development of age-associated pathologies. Consequently, many postmenopausal women undergo estrogen hormone therapy (HT) or self- medicate with over-the counter DHEA (an estrogen precursor), even though the underlying neuroendocrine mechanisms are poorly understood and the long-term effects of these hormonal manipulations on the central nervous system (CNS) are unclear, especially in women who are obese. Using the rhesus macaque (Macaca mulatta), we previously showed that long-term administration of estradiol to old animals could significantly improve cognitive performance, and that perturbed 24-hour activity-rest cycles in aged individuals were associated with inferior cognitive performance as well as compromised immune response. Importantly, the beneficial effects of estradiol on various physiological functions were not sustained if the animals were maintained on a high-fat Western-style diet (WSD). Our overall goal during the next 4 years of study is to use modern gene expression and epigenetic profiling approaches and MR imaging techniques to gain insights into the underlying mechanisms by which menopausal hormonal changes negatively impact the CNS, and to evaluate the efficacy of HT at alleviating or reversing these effects. The proposed studies will be performed entirely in vitro, using previously archived brain tissue from the following groups of female rhesus macaques: 1. Young adult ovary-intact females (on a standard diet) 2. Old ovary-intact females (on a standard diet) 3. Old ovariectomized females (on a standard diet) 4. Old ovariectomized females (on a standard diet + treated with HT for ~3 years) 5. Old ovariectomized females (on a WSD for ~3 years) 6. Old ovariectomized females (on a WSD for ~3 years + treated with HT for ~3 years) Aim 1 will use RNAseq to profile gene expression in the prefrontal cortex, hippocampus, amygdala and cerebellum. Our goal is to elucidate changes within the CNS that correlate with the physiological parameters that we previously examined in vivo, including: age, diet, hormonal status, cognitive performance, circadian activity and sleep quality. We anticipate disclosing significant differences in the expression of genes associated with inflammation, excitatory neurotransmission and synaptic function. Aim 2 will examine epigenetic signals in the same brain areas, while Aim 3 will focus on the analysis of structural and connectivity differences within the CNS, using previously acquired MRI scans. This study will be the first comprehensive profiling of central gene expression changes, epigenetic signals and the connectosome of a highly-translational model of human aging.